Phorboxazoles A and B: Potent Cytostatic Macrolides from Marine

M in the National Cancer Institute's 60 tumor cell line panel). Cytotoxic macrolides have been isolated from a variety of sponge species. Recent examp...
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J. Am. Chem. SOC. 1995,117, 8126-8131

Phorboxazoles A and B: Potent Cytostatic Macrolides from Marine Sponge Phorbas Sp.t Philip A. Searle and Tadeusz F. Molinski* Contribution from the Department of Chemistry, University of Califomia, Davis, Califomia 95616 Received May 2, 1 9 9 9

Abstract: Two new antifungal and cytostatic macrolides, phorboxazoles A (1) and B (2), have been isolated from the Indian Ocean marine sponge Phorbas sp. Structural assignment was accomplished through extensive 2D NMR spectroscopy including COSY, RCT, HMQC, and HMBC. The complete relative stereochemistry about the macrolide ring and solution conformation of 1 were established from analysis of ROESY experiments. Phorboxazoles A and B exhibit in vitro antifungal activity against Candida albicans at 0.1 pgldisk and extraordinary cytostatic activity (mean GI50 7.9 x M in the National Cancer Institute's 60 tumor cell line panel).

Cytotoxic macrolides have been isolated from a variety of sponge species. Recent examples include the exceedingly potent cytostatic agents halichondrin, spongiastatin-1I (=cinachyrolide2), spongiastatins 2 and 3,3 and altohyrtins A-C and desacetylaltohyrtin The latter four compounds are structural analogs of the "spongopyran" parent skeleton while all compounds bear multiple oxane and spiroketal-oxane rings.' In our study of antifungal compounds from marine invertebrates, we examined an extract of the sponge Phorbas sp. which exhibited antifungal activity against Candida albicans. Bioassay-guided separation of the extract afforded two novel isomeric antifungal macrolides, phorboxazoles A (1) and B (2). Like R.

been assigned relative configuration. This is only the second report of natural products from the genus Phorbas.'

Results The methanol extract of the sponge (236 g dry weight) was subjected to a modified Kupchan solvent partition,8 and the CHCl3 fraction was further separated by flash chromatography (silica gel), c18 reversed-phase chromatography, and HPLC (Microsorb c18) to afford phorboxazole A (1; 95.1 mg, 0.040% dry weight) and phorboxazole B (2; 40.5 mg, 0.017%), both as pale yellow amorphous solids. Phorboxazole A. The molecular formula of C53H71N2013Br for 1 was established by HRFABMS (m/z 1023.4243, MH+, A 2.5 m u ) . The UV spectrum contained several overlapping bands at A 235 nm (log E 4.9) indicative of conjugation. The 'H NMR spectrum in CDCl3 displayed well-resolved signals (Figure 1). Analysis of the COSY, long-range COSY, 'H-IH relayed coherence transfer (RCT), HMQC,9 and HMBC'O spectra gave substructures A-E.

the spongiastatins, compounds 1 and 2 show exceptional cytostatic activity; however, they are the first members of a new class of macrolides with an unprecedented carbon skeleton. The phorboxazole skeleton contains 7 rings, including 2 oxazoles and 4 oxane rings, and 15 stereogenic centers, 13 of which have

* To whom correspondence should be addressed. Tel.: (916) 752-6358. FAX: (916) 752-8995. ' In memory of Matt Suffness. National Cancer Institute, 12/15/42 to 6/ 14/95. Abstract published in Advance ACS Abstracts, July 15, 1995. (1) Pettit, G.R.; Cichacz, Z. A.; Gao, F.; Herald, C. L.; Boyd, M. R.; Schmidt, J. M.; Hooper, J. N. J. Org. Chem. 1993, 58, 1302-1304. (2) Fusetani, N.; Shinoda, K.; Matsunaga, S. J. Am. Chem. Soc. 1993, 115, 3977-3981. (3) Pettit, G.R.; Cichacz, Z . A,; Gao, F.; Herald, C. L.; Boyd, M. R. J. Chem. Soc., Chem. Commun. 1993, 1166- 1168. (4) Kobayashi, M.; Aoki, S.; Sakai, H.; Kawazoe, K.; Kihara, N.; Sasaki, T.; Kitagawa, I. Tetrahedron Lett. 1993, 34, 2795-2798. (5) Kobayashi, M.; Aoki, S.; Sakai, H.; Kihara, N.; Sasaki, T.; Kitagawa, I. Chem. Pharm. Bull. 1993, 41, 989-991. (6) Kobayashi, M.; Aoki, S.;Kitagawa, I. Tetrahedron Lett. 1994, 35, 1243- 1246. @

D o

o

E o

C2-Cl8: Substructure A. The largest substructure, A, was highly oxygenated and delineated by COSY and relayed coherence transfer (RCT) experiments. The IH NMR signals for the olefinic protons H2 and H3 were unresolved in CDCl3, appearing as a multiplet at d 5.90; however, in CDsOD, H2 (7) Rudi, A.; Stein, Z.; Green, S.;Goldberg, I.; Kashman, Y.: Benayahu, Tetrahedron Lett. 1994, 35, 2589-2592. (8)Kupchan, S. M.; Britton, R. W.; Ziegler, M. F.; Sigel, C. W. J. Org. Chem. 1973, 38, 178-179. (9) Bax, A.; Griffey, R. H.; Hawkins, B. L. J. Am. Chem. SOC.1983. 105, 7188-7190. (10) Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 20932094.

Y.;Schleyer, M.

0002-786319511517-8126$09.00/00 1995 American Chemical Society

Phorboxazoles A and B from Marine Sponge Phorbas Species

1.0

3.5

3.0

u

20

1,s

1.0

SJ

s.0

4.S

ppm

7s

7.0

6.0

ppm

Figure 1. 'H NMR spectrum of phorboxazole A (l),CDC13,500MHz.

and H3 were readily distinguished (6 5.96, ddd, J = 11.3, 3.0, 1.0, H2; 6.07, ddd, J = 11.3, 9.2, 3.5, H3) and showed a relatively small J2.3 coupling (1 1 Hz) consistent with a (Z)-olefin. The COSY spectrum (CDC13) showed the signal at 6 5.90 to be coupled to both of the highly anisochronous methylene protons H4a,b (6 2.30, m; 3.46, m) which coupled in sequence to an oxymethine H5 (6 4.15) and methylene protons H6a,b (6 2.04, br d, J = 13.2 Hz; 2.40, m). The H6 proton signals were allylically coupled to an exocyclic methylene group H5la,b (6 4.60, br s; 4.98, br s) which was flanked by another methylene H8a,b (6 1.86, m; 2.69, br d, J = 11.9 Hz). The sequence C8C15 was comprised of a tetrad of alternating methylene and oxymethine groups, terminated by a 2,4-disubstituted oxazole, the latter evidenced by the presence of benzylic coupling ( J < 1 Hz) between H15 (6 4.72, dd, J = 10.8, 2.9 Hz) and an oxazole signal H17 (6 7.40, s, 'Jc-H= 205 Hz). C19-C26: Substructure B. Determination of the segment inclusive of H20 to H26 was made by combined analysis of COSY, RCT, and HMBC spectra. The protons H19 and H20 comprised a disubstituted (@-olefin (JI9.20 = 16.0 Hz), with H20 sequentially coupled to methylene protons H21 (6 2.40, m, 1H; 2.51, ddd, IH, J = 12.7,9.7,5.3 Hz) and an oxymethine H22 (6 3.52) which in tum correlated to a methine proton H23 at 6 2.30. H23 is in turn coupled to a methyl doublet (6 0.95) and an oxygenated methine H24 (6 4.49). Although the H23 signal (6 2.30) was severely overlapped by other signals, RCT correlations were observed from H22 to the methyl doublet signal H50 (6 0.95, d, J = 6.8 Hz) and from H24 (6 4.49, dd, J = 11.2,4.4 Hz) to H50. H24 was further coupled to a methine proton H25 (6 2.01) and the latter Correlated to another methyl doublet signal H49 (6 0.75, d, J = 6.4 Hz) and an oxymethine H26 (6 3.56). This connection was supported by RCT correlations H49-H24 and H49-H26. C27-C31: Substructure C. In the COSY spectrum the olefinic proton H28 (6 6.22, bs) showed fine long range coupling (J 1Hz) to both a vinylic methyl group H48 (6 1.96, br s) and a second oxazole singlet H30 (6 7.56, s, = 208 Hz). The C48 chemical shift (6 14.19, q) was consistent with an (E)-trisubstituted olefin.' I C34-C40: Substructure D. In the COSY spectrum, the C34 methylene protons (6 1.34, 2.30) were correlated to an oxygenated methine (6 3.74) which was in tum coupled to a methylene at C36 (6 1.07, 1.95). These methylene protons were

J. Am. Chem. SOC.,Vol. 117, No. 31, 1995 8127 in tum correlated to an oxygenated methine H37 (6 3.79) which coupled to another oxygenated methine H38 (6 4.30). H38 coupled to an olefinic proton (6 5.34) which showed allylic coupling to a methyl group (6 1.79). (@-Geometry for the C39-C40 olefin was implied by the upfield I3C chemical shift for the vinyl methyl group (C47, 6 13.42, q)." C41-C46: Substructure E. Connectivities from C41 to C46 were revealed by examination of the COSY spectrum. (E)Olefinic protons H42 and H43 (J41.42 = 15.7 Hz) were coupled to an oxymethine (6 3.63) which was sequentially connected to methylene protons H44 (6 2.24, 2.30) followed by vinyl protons H45 (6 6.15, ddd) and H46 (6.07, ddd) of an (,?)-olefin (J45.46 = 13.6 Hz). The I3C chemical shift for C46 (6 106.35) and comparison with model compoundsI2 allowed placement of the bromine at C46 as a terminal vinyl bromide. It is noteworthy that C46, C45, and C44 showed relatively intense I3C NMR signals due to enhanced scalar relaxation through the nearby 79Br/81Brn u ~ l e u s . ' ~ . ' ~ Assembly of Substructures. Assembly of the substructures through nonprotonated carbons was established through the HMBC data (Table l).I5 An a$-unsaturated ester (v 1718 cm-I) was located by correlation of H2 and H3 (overlapped, 6 5.90, m, 2H) to carbonyl group C1 (6 165.58, s), a methylene group C4 (6 30.44, t) and oxymethine C5 (6 73.45, d). Fragment C5 to C15 was accommodated, on the basis of HMBC correlations H5-C9 and H15-Cll, by two oxane rings joined by a methylene group at C10. The oxazole proton H17 (6 7.40, s) showed a correlation to both C16 and C18, and with assignment of these two carbons, comparison with I3C data for other substituted oxazoles,16in particular one-bond JCH coupling constants," confirmed a second 2,4-disubstituted oxazole group. The downfield oxazole signal C18 (6 161.28, s) showed longrange heteronuclear correlation (HMBC) to E-disubstituted vinyl group signals H19 (6 6.28, d, J = 16.0 Hz) and H20 (6 6.66, ddd, J = 16.0, 9.7, 6.4 Hz) and allowed attachment of substructures A and B as shown. Extensive HMBC correlations from H26 to C27, C28, and C48 and from H48 to C26 connected partial structures B and C. The second oxazole proton signal H30 showed HMBC correlations to C29 and C31, consistent with a 2,4-substituted oxazole while C31 correlated to an isolated methylene group H32a,b (AB quartet, 6 3.08, 3.14, J,,, = 15.4 Hz). In turn, H32a,b correlated to a quaternary carbon at 6 96.61 assigned to the hemiketal carbon C33 which was linked to adjacent methylene protons H34a,b, thus linking substructures C with D. The final substructure was attached via two- and three-bond HMBC correlations from H47 to C39, C40, and C41 and from H41 to C39 and C47. Three oxane rings were located within the fragment C5-C26. 'H-IH coupling constants were consistent with the axial and equatorial couplings of one trans- and two cis-fused oxane rings at C5C9, Cll-Cl5, and C22-C26, respectively (see Stereochemistry section). The three oxane rings were confirmed by observation of HMBC correlations from H5 to C9 and from H15 to C11 and a weak correlation from H26 to C22. The fourth oxane (11) Couperas, P. A,; Clague, A. D. H.; Dongen, J. P. C. M. Org. Mugn. Reson. 1976. 8. 426-431. (12) Patil, A. D.; Kokke, W. C.; Cochran, S . ; Francis, T. A,; Tomszek, T.; Westley, J. W. J . Nut. Prod. 1992, 55, 1170-1177. (13)Nohon, R. S . ; Croft, K. D.; Wells, R. J. Tetrahedron 1981, 37, ~~

2341-2349. (14) Wehrli, F. W.; Marchand, A. P.;Wehrli, S . Interpretation of Curbon13 NMR Spectra; Wiley: Chichester, 1983; pp 58-62. (15) For the assignment of nonequivalent methylene protons, Ha refers to the higher field proton, while Hb refers to the lower field proton. (16) Adamczeski, M.; Quifioa, E.; Crews, P. J. Am. Chem. Soc. 1988, 110, 1598-1602. (17) Maryanoff, C. A. In The Chemistry of Heterocyclic Compounds; Turchi, I. J., Ed.; Wiley: New York, 1986; Vol. 45, pp 343-360.

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Searle and Molinsti

Table 1. 'H NMR and 13C NMR Chemical Shifts8 and HMBC and ROESY Correlations* of Phorboxazole A (1) no. 1 2 3 4a 4b 5 6a 6b 7 8a 8b 9 10a 10b

11 12a 12b 13 14a,b 15 16 17 18 19 20 21a 21b 22 23 24 25 26 27 28 29 30 31 32a 32b 33 34a 34b 35 36a 36b 37 38 39 40 41 42 43 44a 44b 45 46 47 48 49 50 51a 51b 35-OMe 43-OMe 33-OH

I3C NMR 6 (mult) 165.58 (s) 120.97 (d) 144.40 (d) 30.44 (t) 73.45 (d) 36.92 (t) 141.66 (s) 38.94 (t)" 69.10 (d) 41.24 (t) 68.60 (d) 38.96 (t)" 64.28 (d) 34.93 (t) 66.91 (d) 142.08 (s) 133.71 (d)b 161.28 (s) 119.29 (d) 134.07 (d) 34.32 (t) 77.98 (d) 32.50 (d) 79.28 (d) 3 1.67 (d) 89.15 (d) 137.94 (s) 118.47 (d) 137.48 (s)c 135.89 (d) 160.03 (s) 39.71 (t) 96.61 (s) 40.40 (t) 73.00 (d) 33.02 (t) 72.45 (d) 70.85 (d) 129.87 (d) 137.50 (s)c 136.96 (d) 128.87 (d) 81.06 (d) 39.18 (t) 133.66 (d)b 106.35 (d) 13.42 (4) 14.19 (4) 13.28 (4) 5.98 (4) 110.08 (t) 55.74 (4) 56.34 (4)

'H NMR 6 (mult, J (Hz), (int) 5.90 (m, 1H) 5.90 (m, 1H) 2.30 (m, 1H) 3.46 (m, IH) 4.15 (brddd, 11.9,5.0,5.0, 1H) 2.04 (br d, 13.2, 1H) 2.40 (m, 1H) 1.86 (m, 1H) 2.69 (br d, 11.9, 1H) 3.96 (dddd, 10.4, 10.2, 5.1, 2.5, 1H) 1.41 (ddd, 13.2, 10.2, 3.1, IH) 1.86 (m, 1H) 4.05 (brddd, 11.4, 11.4,3.2, 1H) 1.56 (ddd, 13.6, 11.4, 2.7, 1H) 1.68 (bd, 13.6, 1H) 4.36 (m, 1H) 1.95 (m, 2H) 4.72 (dd, 10.8, 2.9, 1H)

HMBC correlations C1, C4 c1, c4, c 5 c2, c 3 c7, c 9 C7, C8, C5 1

ROESY correlation

H4a, H5, H6a H3, H4b, H5 H4a, H9 H3, H4a, H6a, H6b H3, H5, H6b, H51a H5, H6a, H8a

C11, C13, C16, C17

H6b, H8b H8a,H9,H11,H51b H4b, H8b, HlOb HlOb, H11 H9, HlOa, H12a H8b, HlOa, H12b, HI5 HlOb, H12b, H13 H11, H12a, H13 H12a. H12b, H14 H13, H15, H17 HI 1, H14

7.40 (s, l'H)d

C16, C18

HI4

6.28 (d, 16.0, 1H) 6.66 (ddd, 16.0,9.7, 6.4, 1H) 2.40 (m, 1H) 2.51 (ddd, 12.7,9.7, 5.3, 1H) 3.52 (ddd, 11.1,5.3, 1.0, 1H) 2.30 (m, 1H) 4.49 (dd, 11.2,4.4, 1H) 2.01 (m, IH) 3.56 (d, 10.1, 1H)

C18, C21 C18. C21 C19, C20 C50 C1, C49, C50 C24, C26 C22, C27, C28, C48

H22, H50 H21a, H21b, H22, H23, H50, H51b H20, H2 1b, H50 H20, H2 1a, H22 H19, H20, H21b, H23, H24 H20, H22, H24, H50 H22, H23, H26, H49 H49, H50 H24, H28, H49

6.22 (s, 1H)

C26, C27, C30, C48

H26, H30, H49

7.56 (s, 1H)'

C29, C3 1

H28, H48

3.08 (d, 15.4, 1H) 3.14 (d, 15.4, 1H)

C31, C33 C31. C33

H34a, 33-OH H34a. 33-OH

1.34 (ddd, 11.8, 11.8, 1.6, 1H) 2.30 (m, 1H) 3.74(dddd, 11.8, 11.8,4.6,4.6, 1H) 1.07 (ddd, 12.0, 11.8, 11.8, 1H) 1.95 (m, 1H) 3.79 (ddd, 12.0,7.9, 2.0, 1H) 4.30 (dd, 9.0, 7.9, 1H) 5.34 (d, 9.0, 1H)

c33, c 3 5

C38, C39 C37, C39, C40 C37, C41, C47

H32a, H32b. H34b, H36a H34a, H35,35-OMe H34b, H36b, 35-OMe H34a, H36b, H38 H35, H36a, H37.35-OMe H36b, H47,33-0H H36a, H4 1, H47 H41, H47

c39, c47 C40, C43, C44 C41, C44, C45, OMe C42, C43, C45, C46

H38, H39, H47,43-OMe H44a, H47,43-OMe H47,43-OMe H42

6.17 (d, 15.7, 1H) 5.48 (dd, 15.7,7.9, 1H) 3.63 (ddd, 7.9.6.0, 5.7, 1H) 2.24 (dddd, 14.5, 7.1, 5.7, 1.1, 1H) 2.30 (m, 1H) 6.15 (ddd, 13.6, 7.1, 7.1, 1H) 6.07 (ddd, 13.6, 1.1, 1.1, 1H) 1.79 (s, 3H) 1.96 (s, 3H) 0.75 (d, 6.4, 3H) 0.95 (d, 6.8, 3H) 4.60 (br s, 1H) 4.98 (br s, 1H) 3.34 (s, 3H) 3.24 (s, 3H) 5.31 (s, IHY

C6, C7, C5 1 c9

c11 C13, C14 C11, C15

C35, C38

C43, C46 c44,c 4 5 C39, C40, C41 C26, C27, C28 C24, C25, C26 C22, C23, C24 C6, C8 C6, C8 c35 c43

H37, H38, H39, H41, H42, H43 H30 H24, H25, H26, H28 H19, H20, H21a, H23, H25, H51a, H51b H6a, H50, H5 1b H8b, H20, H50, H5 la H34b, H35, H36b H41, H42, H43 H32a. H32b. H37

a-c Assignments interchangeable. 'JCH= 205 Hz. E 'JCH = 208 Hz. f Remaining OH signals; 2.10 (br s, lH), 2.71 (br s, IH). g Recorded in CDCI, at 500 MHz ('H) and 100 MHz (I3C). HMBC optimized for 2 , 3 J = ~ ~10 Hz. ROESY mixing time tm 500 mS.

ring was revealed by NOE from 33-OH to H37 across the oxygen bridge (see stereochemistry). The positions of the two methoxyl groups were located on C43 and C35 on the basis ofHMBC correlations to these carbons, leaving two free hydroxyl groups at C13 and C38. Finally, HMBC showed a three-bond correlation beween the ester oxymethine H24 (6 4.49,

dd) and carbonyl C1 (6 165.58, s), thus closing the macrolide ring. Attempts to acetylate 1 (AczO, py, 23 "C) to confirm the hydroxyl group placements resulted in extensive decomposition of the molecule. Stereochemistry. Relative stereochemistry was determined through analysis of 'H-IH coupling constants and ROESY data

J. Am. Chem. SOC.,Vol. 117, No. 31, 1995 8129

Phorboxazoles A and B from Marine Sponge Phorbas Species

........_ H5 la]

1.0

1

v

4.b

4.1

1.:

1

0

-

1.0

Figure 2. Summary of ROESY correlations observed for phorboxazole A (1). f m 500 mS, CDC13. Geminal and some vicinal correlations omitted

4.9

0

4.8

4.1

4.6

4.1

4.7

4.6

for clarity. (Figure 2) and was analyzed independently for each molecular fragment and finally linked to provide configurational assignment about the large ring. (a) C33-C37 Fragment. A large coupling (12.0 Hz) between H37 and H36a indicated a diaxial arrangement for these protons. H35 was also assigned as axial on the basis of large couplings (1 1.8 Hz) to both H34a and H36a. The ketal hydroxyl group was established as being in an axial position due to observation of a small w-coupling to H34a and a ROESY correlation to H37. (b) C22-C26 Fragment. A large coupling (J = 10.1 Hz) between H26 and H25 indicated a diaxial arrangement for these protons and an equatorial methyl group (H49). The other methyl group (H50) was assigned as axial due to observation of a ROESY correlation to H25 and the particularly upfield I3C NMR chemical shift for C50 (6 5.98).14 H24 exhibited a large coupling (1 1.2 Hz) to H25 and ROESY correlations to H22 and H26; thus, both H24 and H22 are in axial positions. (c) CS-ClS Fragment. An axial position for H15 was evident from the large coupling (10.8 Hz) to H14 and a ROESY correlation to an axial H1 1, with H11 exhibiting a large coupling (11.4 Hz) to H12a. H13 showed only a small coupling (2.7 Hz) to H12a and was assigned an equatorial position. The stereochemistry of the Cll-C15 ring could be correlated to that of the second ring C5-C9 through the methylene bridge C10 (Figure 2). H11 exhibited a large coupling (11.4 Hz) to HlOb and showed a ROESY correlation to only HlOa, indicating an anti arrangement of H11 and HlOb. A large coupling between HlOa and H9 and a ROESY correlation between HlOb and H9 indicated that H9 and HlOa were also in a diaxial arrangement. H9 also exhibited a large coupling (10.2 Hz) to H8a and thus occupies an axial position on the ring. A ROESY correlation between H6b and H8a confirmed axial positions for these two protons, while H6a and H8b showed ROESY correlations to H5 l a and H5 lb, respectively, indicating the chair conformation depicted in Figure 2. H5 occupied an equatorial position, as evident from the small couplings to both H6a and H6b. The axial position of C4 was also confirmed by a ROESY correlation from H4b to H9. (d) Complete Macrolide Ring. Simultaneous assignment of both conformation and relative configuration in novel medium- and large-ring substituted macrolides is seldom possible due to lack of a sufficient number of independent variables and interruption of the carbon chain by quaternary

I ,

~~~

1.0

19

PPm

Figure 3. Expansion of the ROESY spectrum of 1 (t, 500 mS, CDC13)

showing transannular NOEs. Only negative contours shown. carbons. An exception was found in the case of 1 due to the conformational restrictions imposed by the presence of three oxane rings and one oxazole along the perimeter of the macrolide annulus. From examination of coupling constants and NOE studies, compound 1 appears to be a relatively rigid ring. Measurement of NOESY spectra gave few useful cross peaks; however, the ROESY spectrum (500 MHz, CDCl3, tm 500 ms) provided a rich data set of correlations, allowing assignment of the relative stereochemistry between the two sections of the macrolide ring, C5-Cl5 and C22-C26 (Figures 2 and 3). Because the relative configurations intemal to each oxane ring (ax-eq) were determined, it only remained to examine all possible combinations of oxane ring stereochemistries that would fit the NOE constraints. Analysis of Dreiding molecular models revealed, that of the four possible arrangements of the macrolide ring of 1, only the combination of oxane relative stereochemistries depicted in Figure 2 would account for observed ROESY correlations between H5 1 and H50-H20 (contacts < 4 k)and satisfy NOEs observed (H8eq to Hllax) between oxane rings Cll-C15 and C5-C9. All ROESY correlations were in full agreement with the assigned stereochemistry and 3D solution structure. Understandably, the relative stereochemistry between the macrolide ring and the C33-C37 oxane ring could not be established, although vicinal coupling constants and ROESY showed it was cis-substitued at C33 and C37 with an axial hemiketal OH as expected from the anomeric effect. The configurations of C38 and C43 remain unassigned, as does the absolute configuration of 1. The solution structure of the phorboxazole molecule reveals an interesting topology that results from a relatively rigid macrolide ring and a trans-fused C5-C9 oxane ring which places the exocyclic methylene C51 directly above the center of the macrolide ring. The molecule thus adopts a “capped annulus” or bowl-shaped conformation with entry to the macrolide ring blocked from one face. Phorboxazole B. Phorboxazole B (2, C53H71N2013Br)is isomeric with 1 (ndz 1023.4271, MH+, A 6.3 m u ) . Com-

8130 J. Am. Chem. SOC., Vol. 117, No. 31, 1995

Searle and Molinski

parison of the IH NMR and I3C NMR spectra of phorboxazole B (2) showed almost identical features. The only chemical shift differences were observed in substructure A. Careful analysis of 'H vicinal coupling constants of protons neighboring H13 revealed that the multiplet pattern of H13 was now different and included two diaxial vicinal couplings (H14a, 6 1.63, br ddd, J = 11.6, 11.6, 11.6 Hz), consistent with an equatorial C13 hydroxyl group. ROESY correlations were observed from H13 to H15ax and Hllax, but otherwise the ROESY map of 2 was identical to that of 1. Therefore, phorboxazole B is the C13 epimer of 1.

compounds gave mean GIso's < 7.9 x M; however, most cell lines were still 100% inhibited at this, the lowest, test concentration. Compounds 1 and 2 thus show activity on the same order of magnitude reported for spongistatin-1' and are among the most potent cytostatic agents yet discovered. Pettit has commented on the empirical correlation of molecular structures bearing multiple oxane and spiroketal rings in macrolides and polyethers from marine invertebrates with exceptional cell growth inhibitory activity.32 Further biological evaluation of 1 and 2 is in progress, and full results will be reported in due course.

Discussion

Experimental Section

Only one report has appeared describing natural products from the genus Phorbas, four phenylpyrrolyloxazoles named phorbazoles A-D.7 A steadily increasing family of oxazolecontaining compounds have been isolated from marine invertebrates over the past decade. For example, the molecular structures of ulapualides A and B,Is kabiramides B and C,I9 halich~ndramides,'~ mycalolides,2° and jaspisamides*' isolated from sponges or the nudibranch Hexabranchus sanguineus each contain a distinctive contiguous trisoxazole moiety. Less frequently encountered bisoxazoles are represented by bengazoles A and B, reported by Crews et a1.,l6 bengazoles B1 and C-G reported by our group,22several related C10 desoxybeng a z ~ l e s , *and ~ . ~hennoxazole, ~ reported by Scheuer et al.25and recently synthesized by Wipf and co-workers.26 The theonezolides A-C2'-** are interesting macrolides containing a thiazole-oxazole combination. Although kabiramide and theonezolide classes of macrolides also embody oxazole units, the phorboxazoles are structurally unrelated and compounds 1 and 2 represent a new class of macrolides containing an unprecedented oxazole-trisoxane fused ring system. Biological Activity. Compounds 1 and 2 exhibited potent activity against C.albicans, but showed no ergosterol depend e n ~ e . * Phorboxazole ~ A (1) gave the following zones of inhibition in the agar disk diffusion assay: 12 mm (loading, 1 pg) and 9 mm (0.1 pg). Phorboxazole B (2) was marginally less active: 11 mm (1 pg) and 8 mm (0.1 pg). Both compounds were also active against Saccharomyces carlsbergensis: 1, 20 mm (1 pg) and 13 mm (0.1 pg); 2, 16 mm (1 pg) and 10 mm (0.1 pg). Neither compound showed antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus in agar disk diffusion assays. Replicate testing of 1 and 2 in the National Cancer Institute's panel of 60 tumor cell line^^^.^' showed exceptional inhibition of cell growth. Both

General proCedures. General procedures can be found elsewhere.33 Mass spectra were provided by the University of Minnesota Chemistry Department Mass Spectrometry Service Laboratory. N M R Spectroscopy. NMR spectra were measured at 300 or 500 MHz ('H) and 75.4, 100, or 125 MHz (I3C). 'H NMR and I3C NMR are referenced to CDC13 solvent signals at 7.26 and 77.00 ppm, respectively. Multiplicities of I3C spectra were assigned by DEPT experiments. Standard pulse sequences were employed for DEPT and magnitude COSY, LRCOSY, RCT, and HETCOR. Phase sensitive ROESY spe'ctra were measured with a mixing time rmof 500 ms, while HMQC and HMBC were optimized for 'Jc-H =140 Hz and 2 - 3 J ~ = -~ 10 Hz, respectively. Isolation. The sponge (93-05-054) was collected in January 1993 by hand using SCUBA near Muiron Island (21' 40' S , 114' 18' E), Westem Australia. The animals were immediately frozen at -20 "C until required. The sponge sample, described by John Hooper, Queensland Museum, Brisbane, Australia, is a new species of Phorbus (see supporting information). A voucher specimen is archived at the Queensland Museum (QM G304923). Lyophilized animals (236 g) were extracted with MeOH (3 x 2 L) and filtered. The extracts were combined, concentrated to approximately 300 mL, and successively extracted using a modified Kupchan partition8 as follows. The water content (% vlv) of the MeOH extract was adjusted prior to sequential partitioning against n-hexane (10% v/v HzO), CCL (20%), and CHCl3 (40%). The aqueous phase was concentrated to remove MeOH and then extracted with n-BuOH. The CHC13 (2.19 g) extract was fractionated by flash chromatography on silica gel (stepwise gradient elution, CHCIJMeOH (99:l) to 100% MeOH), followed by reversedphase chromatography (CISsilica cartridge, MeOWHzO (9: 1)) and HPLC (Microsorb C18 column, 10 x 300 mm, MeOWH2O (82:18)) to afford 1 (95.1 mg, 0.040% dry weight of animal) and 2 (40.5 mg, 0.017%). both as pale yellow amorphous solids. Data for Phorboxazole A (1): C53H71N2013Br;[ a ]+44.8" ~ (c 1.0 MeOH); UV (MeOH) A,, 235 nm (log E 4.9); IR (film) vmax3390 (br), 1718, 1188, 1158, 1089,731 cm-I; 'Hand I3C NMR see Table 1; FABMS d z 1025 (MH', 8'Br, 100%), 1023 (MH+, 79Br, 100%), 1007 (MH+ - H20, 8'Br,57%), 1005 (MH' - H20, 79Br,55%); HRFABMS found d z 1023.4243 (MH+), C53H72N~013Brrequires 1023.4218. Data for Phorboxazole B (2): C53H71N2013Br;[ a ] +44.4" ~ (c 1.0 MeOH); UV (MeOH) A,, 235 nm (log E 4.8); IR (film) umaX 3390 (br), 1718, 1192, 1158, 1089, 731 cm-I; 'H and I3C NMR see Table 1; FABMS m/z 1025 (MH', loo%), 1023 (MH+, 79Br, 100%). 1007 (MH+ - H20, 8'Br, 43%). 1005 (MH+ - HzO, 79Br,45%); HRFABMS found n d z 1023.4271 (MH+), C53H72N2013Br requires 1023.4218.

(18) Roesener, J. A.; Scheuer, P. J. J. Am. Chem. SOC. 1986, 108, 846847. (19) Matsunaga, S.; Fusetani, N.; Hashimoto, K.; Koseki, K.; Noma, M. J . Am. Chem. SOC. 1986, 108, 847-849. (20) Fusetani, N.; Yasumuro, K.; Matsunaga, S . ; Hashimoto, K. Tetruhedron Lett. 1989, 30, 2809-2812. (21) Kobayashi, J.; Murata, 0.;Shigemon, H. J. Nut. Prod. 1993, 56, 787-791. (22) Molinski, T. F. J. Nut. Prod. 1993, 56, 1-8. (23) Rodriguez, J.; Nieto, R. M.; Crews, P. J. Nut Prod. 1993,56,20342040. (24) Rudi, A.; Kashman, Y.;Benayahu, Y.; Schleyer, M. J. Nut. Prod. 1994, 57, 829-832. (25) Ichiba. T.: Yoshida. W. Y.: Scheuer, P. J.: Higa. T.: Gravalos. D. G. J. Am. Chem. SOC. 1991, 113, 3173-3174. (26) Wipf, P.; Lim, S. J. Am. Chem. SOC. 1995, 117, 558-559. (27) Kobayashi, J.; Kondo, K.; Ishibashi, M.; Wdchli, M. R.; Nakamura, T. J. Am. Chem. SOC. 1993, 115, 6661-6665. (28) Kondo, K.; Ishibashi, M.; Kobayashi, J. Tetrahedron 1994, 50, 8355-8362. (29)Antonio, J.; Molinski, T. F. J. Nat. Prod. 1993, 56, 54-61. (30) Suffness, M.; Newman, D. J.; Snader, K. In Bioorganic Murine Chemistry; Scheuer, P. J., Ed.; Springer-Verlag: Berlin, 1989; Vol. 3, p 175. 1

Acknowledgment. This work was supported by the National Institutes of Health (Grant AI 31660). We are most grateful to John Hooper (Queensland Museum, Brisbane, Australia) for identification of the sponge, Mary K. Harper (Scripps Institution (31) Boyd, M. R.; Paull, K. D.; Rubinstein, L. R. In Antitumor Drug Discovety and Development; Valenote, F., Corbett, T., Baker, L., Eds.; Kluwer Academic: Amsterdam, 1991; pp 11-34. (32) Pettit, G. R.; Tan, R.; Gao, R. T.; Williams, M. D.; Doubek, D. L.; Boyd, M. R.; Schmidt, J. M.: Chapuis, J.-C.; Hamel, E.; Bai, R.; Hooper, J. N. A.; Tackett, L. P. J. Org. Chem. 1993, 58, 2538-2543. (33) Searle, P. A.; Molinski, T. F. J. Org. Chem. 1993, 58, 7578-7580.

Phorboxazoles A and B from Marine Sponge Phorbas Species

J. Am. Chem. SOC.,Vol. 117, No. 31, 1995 8131

of Oceanography) for taxonomic assistance, Ed Larka, University of Minnesota, Department of Chemistry for FABMS, and Steven Taylor and Denise C. Manker for assistance with sponge collection. We also thank Jeff S. de Ropp for assistance with the 500 MHz NMR spectra. The 500 MHz NMR spectrometer was partially funded through NIH Grant ISIO-RRO4795 and NSF Grant BBS88-04739.

of 1 and 2 and the RCT and HMBC spectra of 1, a table giving complete chemical shift assignments of 2, and text giving the taxonomic description of the sponge sample (16 pages). This material is contained in many libraries on microfiche, immediately follows this article in the microfilm version of the journal, can be ordered from the ACS, and can be downloaded from the Internet see any current masthead page for ordering information and Internet access instructions.

Supporting Information Available: Figures showing the 'H, I3C, DEFT, COSY, HETCOR, HMQC and ROESY spectra

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